The long-term objective of this research project is to understand how cells identify and recycle inactive ribosomes during protein synthesis. Not all ribosomes that initiate translation are able to complete synthesis of full-length proteins. In many instances, incomplete protein synthesis is due to non-stop mRNAs, which are truncated transcripts lacking in-frame stop codons. In bacteria, ribosomes trapped at the 34-ends of non-stop mRNA are rescued by the tmRNA (for transfer-messenger RNA) quality control system. Although tmRNA is ubiquitous throughout the eubacteria, it can be deleted from Escherichia coli cells due to the presence of a parallel tmRNA-independent ribosome rescue pathway. However, both pathways cannot be disrupted in E. coli, demonstrating that ribosome rescue is an essential function for these and probably all cells. This proposal focuses on three fundamental aspects of translational quality control that revolve around ribosome rescue. First, we seek to determine the functional relationship between A-site mRNA cleavage and ribosome rescue. A- site cleavage is a novel RNase activity that truncates mRNA in the A-site codon of stalled ribosomes, thereby producing non-stop mRNA. Biochemical and molecular genetic approaches will be used to identify the A-site nuclease. Identification of the A-site nuclease is necessary to determine whether the activity plays a functional role in either tmRNA-mediated or tmRNA-independent ribosome rescue. Second, we will characterize the recently identified tmRNA-independent rescue pathway, which appears to be mediated by YhdL. The biochemical function and regulation of YhdL will be investigated using in vitro and in vivo approaches. The spectrum of YhdL ribosome rescue activity will be identified, and its role in nascent chain release determined. Finally, the role of DnaK in tmRNA-mediated ribosome rescue will be mechanistically defined. DnaK activity will be ablated using molecular genetic and pharmacological approaches and the effects on tmRNA tagging assessed. A defined in vitro translation system will be used to determine whether DnaK exerts its effects by virtue of its co-translational chaperone activity.